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Title: The expression of the open reading frame of "Arabidopsis" CAX1, but not its cDNA, confers metal tolerance in yeast

Author
item SHIGAKI, TOSHIRO - Children'S Nutrition Research Center (CNRC)
item MEI, HUI - Children'S Nutrition Research Center (CNRC)
item MARSHALL, JOY - Prairie View A & M University
item LI, XIANGKAI - Children'S Nutrition Research Center (CNRC)
item MANOHAR, MURLI - Children'S Nutrition Research Center (CNRC)
item HIRSCHI, KENDAL - Children'S Nutrition Research Center (CNRC)

Submitted to: Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2010
Publication Date: 8/26/2010
Citation: Shigaki, T., Mei, H., Marshall, J., Li, X., Manohar, M., Hirschi, K.D. 2010. The expression of the open reading frame of "Arabidopsis" CAX1, but not its cDNA, confers metal tolerance in yeast. Plant Biology. 12(6):935-939.

Interpretive Summary: Manipulation of plant calcium transporters is important to increase the calcium content in fruits and vegetables. Often we first study plant calcium transporters by expressing them in brewer's yeast because this is a simple and rapid way to analyze function. Previously, a particular plant calcium transporter has been considered inactive in yeast and thus could not be studied in detail. Here we have trimmed this seemingly inactive protein of regulatory elements that now make it active in yeast. Our study demonstrates that this modified transporter is the preferred form to study the transporter in yeast. Our findings now allow future characterization of the transporter using the simple and robust yeast assay system.

Technical Abstract: The biochemical properties and regulation of several plant CAX (CAtion eXchanger)-type vacuolar Ca (2+)/H (+) exchangers have been extensively analyzed in yeast expression assays. In the present study, we compare and contrast the phenotypes of yeast cells expressing the CAX1 cDNA and open reading frame (ORF). We report that the CAX1 ORF, but not the cDNA containing the 3'-untranslated region (UTR), was able to confer Ca (2+) tolerance when expressed in a Ca (2+)-sensitive yeast mutant. Additionally, only yeasts expressing the N-terminal truncated CAX1 ORF were able to grow on high Mn (2+) media, suggesting that removal of the 3'-UTR altered activity. However, removal of the 3'-UTR from another CAX did not alter the yeast phenotypes. Expression studies demonstrated that expressing the CAX1 ORF in yeast elevates CAX1 RNA and protein levels. Our results suggest that the 3'-UTR modulates expression of CAX1 in yeast.